Tremendous commercial potential exists for producing oxaloacetate-derived biochemicals via aerobic or anaerobic bacterial fermentation processes. Aerobic fermentation processes can be used to produce oxaloacetate-derived amino acids such as asparagine, aspartate, methionine, threonine, isoleucine, and lysine. Lysine, in particular, is of great commercial interest in the world market. Raw materials comprise a significant portion of lysine production cost, and hence process yield (product generated per substrate consumed) is an important measure of performance and economic viability. The stringent metabolic regulation of carbon flow (described below) can limit process yields. Carbon flux towards oxaloacetate (OAA) remains constant regardless of system perturbations (J. Vallino et al., Biotechnol. Bioeng., 41, 633-646 (1993)). In one reported fermentation, to maintain this rigid regulation of carbon flow at the low growth rates desirable for lysine production, the cells converted less carbon to oxaloacetate, thereby limiting the lysine yield (R. Kiss et al., Biotechnol. Bioeng., 39, 565-574 (1992)). Hence, a tremendous opportunity exists to improve the process by overcoming the metabolic regulation of carbon flow.
Anaerobic fermentation processes can be used to produce oxaloacetate-derived organic acids such as malate, fumarate, and succinate. Chemical processes using petroleum feedstock can also be used, and have historically been more efficient for production of these organic acids than bacterial fermentations. Succinic acid in particular, and its derivatives, have great potential for use as specialty chemicals. They can be advantageously employed in diverse applications in the food, pharmaceutical, and cosmetics industries, and can also serve as starting materials in the production of commodity chemicals such as 1,4-butanediol and tetrahydrofuran (L. Schilling, FEMS Microbiol. Rev., 16, 101-110 (1995)). Anaerobic rumen bacteria have been considered for use in producing succinic acid via bacterial fermentation processes, but these bacteria tend to lyse during the fermentation. More recently, the strict anaerobe Anaerobiospirillum succiniciproducens has been used, which is more robust and produces higher levels of succinate (R. Datta, U.S. Pat. No. 5,143,833 (1992); R. Datta et al., Eur. Pat. Appl. 405707(1991)).
Commercial fermentation processes use crop-derived carbohydrates to produce bulk biochemicals. Glucose, one common carbohydrate substrate, is usually metabolized via the Embden-Meyerhof-Pamas (EMP) pathway, also known as the glycolytic pathway, to phosphoenolpyruvate (PEP) and then pyruvate. All organisms derive some energy from the glycolytic breakdown of glucose, regardless of whether they are grown aerobically or anaerobically. However, beyond these two intermediates, the pathways for carbon metabolism are different depending on whether the organism grows aerobically or anaerobically, and the fates of PEP and pyruvate depend on the particular organism involved as well as the conditions under which metabolism is taking place.
In aerobic metabolism, the carbon atoms of glucose are oxidized fully to carbon dioxide in a cyclic process known as the tricarboxylic acid (TCA) cycle or, sometimes, the citric acid cycle, or Krebs cycle. The TCA cycle begins when oxaloacetate combines with acetyl-CoA to form citrate. Complete oxidation of glucose during the TCA cycle ultimately liberates significantly more energy from a single molecule of glucose than is extracted during glycolysis alone. In addition to fueling the TCA cycle in aerobic fermentations, oxaloacetate also serves as an important precursor for the synthesis of the amino acids asparagine, aspartate, methionine, threonine, isoleucine and lysine. This aerobic pathway is shown in FIG. 1 for Escherichia coli, the most commonly studied microorganism. Anaerobic organisms, on the other hand, do not fully oxidize glucose. Instead, pyruvate and oxaloacetate are used as acceptor molecules in the reoxidation of reduced cofactors (NADH) generated in the EMP pathway. This leads to the generation and accumulation of reduced biochemicals such as acetate, lactate, ethanol, formate and succinate. This anaerobic pathway for E. coli is shown in FIG. 2.
Intermediates of the TCA cycle are also used in the biosynthesis of many important cellular compounds. For example, xcex1-ketoglutarate is used to biosynthesize the amino acids glutamate, glutamine, arginine, and proline, and succinyl-CoA is used to biosynthesize porphyrins. Under anaerobic conditions, these important intermediates are still needed. As a result, succinyl-CoA, for example, is made under anaerobic conditions from oxaloacetate in a reverse reaction; i.e., the TCA cycle runs backwards from oxaloacetate to succinyl-CoA.
Oxaloacetate that is used for the biosynthesis of these compounds must be replenished if the TCA cycle is to continue unabated and metabolic functionality is to be maintained. Many organisms have thus developed what are known as xe2x80x9canaplerotic pathwaysxe2x80x9d that regenerate intermediates for recruitment into the TCA cycle. Among the important reactions that accomplish this replenishing are those in which oxaloacetate is formed from either PEP or pyruvate. These pathways that resupply intermediates in the TCA cycle can be utilized during either aerobic or anaerobic metabolism.
PEP occupies a central position, or node, in carbohydrate metabolism. As the final intermediate in glycolysis, and hence the immediate precursor in the formation of pyruvate via the action of the enzyme pyruvate kinase, it can serve as a source of energy. Additionally, PEP can replenish intermediates in the TCA cycle via the anaplerotic action of the enzyme PEP carboxylase, which converts PEP directly into the TCA intermediate oxaloacetate. PEP is also often a cosubstrate for glucose uptake into the cell via the phosphotransferase system (PTS) and is used to biosynthesize aromatic amino acids. In many organisms, TCA cycle intermediates can be regenerated directly from pyruvate. For example, pyruvate carboxylase (PYC), which is found in some bacteria but not E. coli, mediates the formation of oxaloacetate by the carboxylation of pyruvate utilizing carboxybiotin. As might be expected, the partitioning of PEP is rigidly regulated by cellular control mechanisms, causing a metabolic xe2x80x9cbottleneckxe2x80x9d which limits the amount and direction of carbon flowing through this juncture. The enzyme-mediated conversions that occur between PEP, pyruvate and oxaloacetate are shown in FIG. 3.
TCA cycle intermediates can also be regenerated in some plants and microorganisms from acetyl-CoA via what is known as the xe2x80x9cglyoxylate shunt,xe2x80x9d xe2x80x9cglyoxylate bypassxe2x80x9d or glyoxylate cycle (FIG. 4). This pathway enables organisms growing on 2-carbon substrates to replenish their oxaloacetate. Examples of 2-carbon substrates include acetate and other fatty acids as well as long-chain n-alkanes. These substrates do not provide a 3-carbon intermediate such as PEP which can be carboxylated to form oxaloacetate. In the glyoxylate shunt, isocitrate from the TCA cycle is cleaved into glyoxylate and succinate by the enzyme isocitrate lyase. The released glyoxylate combines with acetyl-CoA to form malate through the action of the enzyme malate synthase. Both succinate and malate generate oxaloacetate through the TCA cycle. Expression of the genes encoding the glyoxylate bypass enzymes is tightly controlled, and normally these genes are repressed when 3-carbon compounds are available. In E. coli, for example, the genes encoding the glyoxylate bypass enzymes are located on the aceBAK operon and are controlled by several transcriptional regulators: ic/R (A. Sunnarborg et al., J. Bacteriol., 172, 2642-2649 (1990)), fad/R (S. Maloy et al., J. Bacteriol. 148 83-90 (1981)), fruR (A. Chia et al., J. Bacteriol., 171, 2424-2434 (1989)), and arcAB (S. Iuchi et al., J. Bacteriol. 171 868-873 (1989); S. Iuchi et al., Proc. Natl. Acad. Sci. USA, 85, 1888-1892 (1988)). The glyoxylate bypass enzymes are not expressed when E. coli is grown on glucose, glycerol, or pyruvate as a carbon source. The glyoxylate shunt is induced by fatty acids such as acetate (Kornberg, Biochem. J., 99, 1-11 (1966)).
Various metabolic engineering strategies have been pursued, with little success, in an effort to overcome the network rigidity that surrounds carbon metabolism. For example, overexpression of the native enzyme PEP carboxylase in E. coli was shown to increase the carbon flux towards oxaloacetate (C. Millard et al., Appl. Environ. Microbiol., 62, 1808-1810 (1996); W. Farmer et al, Appl. Env. Microbiol., 63, 3205-3210 (1997)); however, such genetic manipulations also cause a decrease in glucose uptake (P. Chao et al., Appl. Env. Microbiol., 59, 4261-4265 (1993)), since PEP is a required cosubstrate for glucose transport via the phosphotransferase system. An attempt to improve lysine biosynthesis in Corynebacterium glutamicum by overexpressing PEP carboxylase was likewise not successful (J. Cremer et al., Appl. Env. Microbiol., 57, 1746-1752 (1991)). In another approach to divert carbon flow toward oxaloacetate, the glyoxylate shunt in E. coli was derepressed by knocking out one of the transcriptional regulators, fadR. Only a slight increase in biochemicals derived from oxaloacetate was observed (W. Farmer et al., Appl. Environ. Microbiol., 63, 3205-3210 (1997)). In a different approach, malic enzyme from Ascaris suum was overproduced in mutant E. coli which were deficient for the enzymes that convert pyruvate to lactate, acetyl-CoA, and formate. This caused pyruvate to be converted to malate which increased succinate production (see FIG. 2). However, this approach is problematic, since the mutant strain in question cannot grow under the strict anaerobic conditions which are required for the optimal fermentation of glucose to organic acids (L. Stols et al., Appl. Biochem. Biotechnol., 63-65, 153-158 (1997)).
A metabolic engineering approach that successfully overcomes the network rigidity that characterizes carbon metabolism and diverts more carbon toward oxaloacetate, thereby increasing the yields of oxaloacetate-derived biochemicals per amount of added glucose, would represent a significant and long awaited advance in the field.
The present invention employs a unique metabolic engineering approach which overcomes a metabolic limitation that cells use to regulate the synthesis of the biochemical oxaloacetate. The invention utilizes metabolic engineering to divert more carbon from pyruvate to oxaloacetate by making use of the enzyme pyruvate carboxylase. This feat can be accomplished by introducing a native (i.e., endogenous) and/or foreign (i.e., heterologous) nucleic acid fragment which encodes a pyruvate carboxylase into a host cell, such that a functional pyruvate carboxylase is overproduced in the cell. Alternatively, the DNA of a cell that endogenously expresses a pyruvate carboxylase can be mutated to alter transcription of the native pyruvate carboxylase gene so as to cause overproduction of the native enzyme. For example, a mutated chromosome can be obtained by employing either chemical or transposon mutagenesis and then screening for mutants with enhanced pyruvate carboxylase activity using methods that are well-known in the art. Overexpression of pyruvate carboxylase causes the flow of carbon to be preferentially diverted toward oxaloacetate and thus increases production of biochemicals which are biosynthesized from oxaloacetate as a metabolic precursor.
Accordingly, the present invention provides a metabolically engineered cell that overexpresses pyruvate carboxylase. Overexpression of pyruvate carboxylase is preferably effected by transforming the cell with a DNA fragment encoding a pyruvate carboxylase that is derived from an organism that endogenously expresses pyruvate carboxylase, such as Rhizobium etli, Corynebacterium glutamicum, Methanobacterium thermoautotrophicum, or Pseudomonas fluorescens. Pyruvate carboxylase can be expressed within the engineered cell from an expression vector, or alternatively from a DNA fragment that has been chromosomally integrated into the cell""s genome. Optionally, the metabolically engineered cell of the invention overexpresses PEP carboxylase in addition to pyruvate carboxylase. Also optionally, the metabolically engineered cell does not express a detectable level of PEP carboxykinase. In a particularly preferred embodiment of the invention, the metabolically engineered cell is a C. glutamicum, E. coli, Brevibacterium flavum, or Brevibacterium lactofermentum cell that expresses a heterologous pyruvate carboxylase.
The invention also includes a method for making a metabolically engineered cell that involves transforming a cell with a nucleic acid fragment that contains a nucleotide sequence encoding an enzyme having pyruvate carboxylase activity, to yield a metabolically engineered cell that overexpresses pyruvate carboxylase. The method optionally includes co-transforming the cell with a nucleic acid fragment that contains a nucleotide sequence encoding an enzyme having PEP carboxylase activity so that the metabolically engineered cells also overexpress PEP carboxylase.
Also included in the invention is a method for making an oxaloacetate-derived biochemical that includes providing a cell that produces the biochemical; transforming the cell with a nucleic acid fragment containing a nucleotide sequence encoding an enzyme having pyruvate carboxylase activity; expressing the enzyme in the cell to cause increased production of the biochemical; and isolating the biochemical from the cell. Preferred biochemicals having oxaloacetate as a metabolic precursor include, but are not limited to, amino acids such as lysine, asparagine, aspartate, methionine, threonine, and isoleucine; organic acids such as succinate, malate and fumarate; pyrimidine nucleotides; and porphyrins.
The invention further includes a nucleic acid fragment isolated from P. fluorescens which contains a nucleotide sequence encoding a pyruvate carboxylase enzyme, preferably the xcex14xcex934 pyruvate carboxylase enzyme produced by P. fluorescens.